1. Field of the Invention
The present invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptides of the present invention have been identified as members of the vascular endothelial growth factor family. More particularly, the polypeptides of the present invention are human vascular endothelial growth factor 2 (VEGF2). The invention also relates to inhibiting the action of such polypeptides. Additionally, the present invention relates to antibodies directed to the polypeptides of the present invention. The present invention also relates to the administration of vascular endothelial growth factor 2 (VEGF-2) polynucleotides and polypeptides to treat disorders of or injuries to photoreceptor cells.
2. Related Art
The formation of new blood vessels, or angiogenesis, is essential for embryonic development, subsequent growth, and tissue repair. Angiogenesis is also an essential part of certain pathological conditions, such as neoplasia (i.e., tumors and gliomas). Abnormal angiogenesis is associated with other diseases such as inflammation, rheumatoid arthritis, psoriasis, and diabetic retinopathy (Folkman, J. and Klagsbrun, M., Science 235:442-447(1987)).
Both acidic and basic fibroblast growth factor molecules are mitogens for endothelial cells and other cell types. Angiotropin and angiogenin can induce angiogenesis, although their functions are unclear (Folkman, J., Cancer Medicine, Lea and Febiger Press, pp. 153-170 (1993)). A highly selective mitogen for vascular endothelial cells is vascular endothelial growth factor or VEGF (Ferrara, N. et al., Endocr. Rev. 13:19-32 (1992)), which is also known as vascular permeability factor (VPF).
Vascular endothelial growth factor is a secreted angiogenic mitogen whose target cell specificity appears to be restricted to vascular endothelial cells. The murine VEGF gene has been characterized and its expression pattern in embryogenesis has been analyzed. A persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, e.g., in choroid plexus and kidney glomeruli. The data was consistent with a role of VEGF as a multifunctional regulator of endothelial cell growth and differentiation (Breier, G. et al., Development 114:521-532 (1992)).
VEGF shares sequence homology with human platelet-derived growth factors, PDGFa and PDGFb (Leung, D. W., et al., Science 246:1306-1309, (1989)). The extent of homology is about 21% and 23%, respectively. Eight cysteine residues contributing to disulfide-bond formation are strictly conserved in these proteins. Although they are similar, there are specific differences between VEGF and PDGF. While PDGF is a major growth factor for connective tissue, VEGF is highly specific for endothelial cells. Alternatively spliced mRNAs have been identified for both VEGF, PLGF, and PDGF and these different splicing products differ in biological activity and in receptor-binding specificity. VEGF and PDGF function as homo-dimers or hetero-dimers and bind to receptors which elicit intrinsic tyrosine kinase activity following receptor dimerization.
VEGF has four different forms of 121, 165, 189 and 206 amino acids due to alternative splicing. VEGF121 and VEGF165 are soluble and are capable of promoting angiogenesis, whereas VEGF189 and VEGF206 are bound to heparin containing proteoglycans in the cell surface. The temporal and spatial expression of VEGF has been correlated with physiological proliferation of the blood vessels (Gajdusek, C. M., and Carbon, S. J., Cell Physiol. 139:570-579 (1989); McNeil, P. L., et al., J. Cell. Biol. 109:811-822 (1989)). Its high affinity binding sites are localized only on endothelial cells in tissue sections (Jakeman, L. B., et al., Clin. Invest. 89:244-253 (1989)). The factor can be isolated from pituitary cells and several tumor cell lines, and has been implicated in some human gliomas (Plate, K. H., Nature 359:845-848 (1992)). Interestingly, expression of VEGF121 or VEGF165 confers on Chinese hamster ovary cells the ability to form tumors in nude mice (Ferrara, N. et al., J. Clin. Invest. 91:160-170 (1993)). The inhibition of VEGF function by anti-VEGF monoclonal antibodies was shown to inhibit tumor growth in immune-deficient mice (Kim, K. J., Nature 362:841-844 (1993)). Further, a dominant-negative mutant of the VEGF receptor has been shown to inhibit growth of glioblastomas in mice.
Vascular permeability factor (VPF) has also been found to be responsible for persistent microvascular hyperpermeability to plasma proteins even after the cessation of injury, which is a characteristic feature of normal wound healing. This suggests that VPF is an important factor in wound healing. Brown, L. F. et al., J. Exp. Med.176:1375-1379 (1992).
The expression of VEGF is high in vascularized tissues, (e.g., lung, heart, placenta and solid tumors) and correlates with angiogenesis both temporally and spatially. VEGF has also been shown to induce angiogenesis in vivo. Since angiogenesis is essential for the repair of normal tissues, especially vascular tissues, VEGF has been proposed for use in promoting vascular tissue repair (e.g., in atherosclerosis).
U.S. Pat. No. 5,073,492, issued Dec. 17, 1991 to Chen et al., discloses a method for synergistically enhancing endothelial cell growth in an appropriate environment which comprises adding to the environment, VEGF, effectors and serum-derived factor. Also, vascular endothelial cell growth factor C sub-unit DNA has been prepared by polymerase chain reaction techniques. The DNA encodes a protein that may exist as either a heterodimer or homodimer. The protein is a mammalian vascular endothelial cell mitogen and, as such, is useful for the promotion of vascular development and repair, as disclosed in European Patent Application No. 92302750.2, published Sep. 30, 1992.
The Retina. The differentiated retina is composed of seven cell types: sensory (rod and cone photoreceptors), glia (Muller cells), and two types of neurons, interneurons, (horizontal, bipolar, and amacrine), and projection neurons (ganglion cells). The development of the various cell types in the retina does not occur synchronously with the majority of the cones, and ganglion and horizontal cells developing before birth. In contrast, differentiation of a majority of the rods, the main cell type in the rat retina, occurs postnatally. Clonal analysis of the progeny of retinal precursor cells has demonstrated that these progenitor cells can produce various combinations of retinal cell types indicating that at least some of the progenitors are multipotential. Furthermore, findings from both in vivo and in vitro studies suggest that the final phenotype of the cell is largely lineage independent which suggest that the changing microenvironment within the retina has a role in determining the cellular potential of the progenitor cells as well as the differentiated phenotype of the progeny.
In vitro, retinal cell proliferation and differentiation is regulated by a variety of factors, for example, FGF-2, CNTF, LIF, TGF, retinoic acid, and BDNF, as well as by extracellular matrix and cell adhesion molecules, for example s-laminin. Yang and Cepko (J. Neurosci. 16(19):6089-6099 (1996)) and more recently Wen et al. (J. Biol. Chem. 273(4):2090-2097(1998)) have identified and characterized the expression pattern of VEGFR-2/FLK-1, a member of the VEGF receptor family. VEGFR transcripts are first detected at E11.5 in association with the developing retinal vasculature and with the central region of the neural retina (Yang and Cepko, J. Neurosci. 16(19):6089-6099 (1996)). Although it is not known if the two events are related, this developmental period is also marked by the onset of ganglion cell development. By developmental day E15, VEGFR-2 expression extends to the periphery of the retina consistent with the outward gradient of retinal development. VEGFR-2 expression was largely localized to the ventricular zone during the perinatal period when neurogenesis is at its peak and a large number of post-mitotic neurons are being formed.
The PDGF/VEGF superfamily currently includes 7 members. The 5 members of the VEGF sub-family bind to 4 different VEGF tyrosine kinase receptors with distinct but overlapping specificities. VEGF, a 34-36 kDa homodimeric glycoprotein that is the prototypic family member, binds to VEGFR-1 and VEGFR-2. VEGF-B and VEGF-D bind only to VEGFR-1 or VEGFR-3, respectively. While VEGF-C, VEGF-2, has the highest affinity for VEGFR-3, it also binds with a lower affinity to VEGFR-2. Once activated the VEGF receptors tyrosine phosphorylate a number of proteins downstream in the signal transduction pathway including phosphatidylinositol 3-kinase, phospholipase C, GAP, and Nck.
The hereditary retinal degenerative diseases (“HRD diseases”) are a group of inherited conditions in which progressive, bilateral degeneration of retinal structures leads to loss of retinal function; these diseases include, for example, age-related macular degeneration, a leading cause of visual impairment in the elderly; Leber's congenital amaurosis, which causes its victims to be born blind; and retinitis pigmentosa (“RP”). RP is the name given to those inherited retinopathies which are characterized by loss of retinal photoreceptors (rods and cones), with retinal electrical responses to light flashes (i.e. electroretinograms, or “ERGs”) that are reduced in amplitude. As the disease progresses, patients show attenuated retinal arterioles, and frequently show “bone spicule” pigmentation of the retina and waxy pallor of the optic discs.
The incidence of RP in the United States is estimated to be about 1:3500 births. Familial cases of RP usually present in childhood with night blindness and loss of midperipheral visual field due to the loss of rods in the peripheral retina. As the condition progresses, contraction of the visual fields eventually leads to blindness. Signs on fundus examination in advanced stages include retinal vessel attenuation, intraretinal pigment in the peripheral fundus, and waxy pallor of the optic disc. Patients have abnormal light-evoked electrical responses from the retina (i.e., electroretinograms or ERGs), even in the early stages in the absence of visible abnormalities on fundus examination. Histopathologic studies have revealed widespread loss of photoreceptors in advanced stages. Therefore, there is a need in the art for methods of treating photoreceptor cell disorders and injuries.